Angiopoietin1 Is Up-regurated In Hip Ligament Of Patient With Ankylosing Spondylitis And Mediates Angiogenesis Through Notch Signaling Pathway

Part ?: Increased matrix degradation and Angiogenesis in AS Hip Joint Ligament TissueBackground: AS as an autoinflammatory disease,inflammation often involving the axial joint and peripheral joints of the tendon ligament attachment point,meanwhile,inflammatory diseases are often associated with vascular regeneration.Hip as the most involvement of the AS in the peripheral large joints,the surrounding ligament is often accompanied with inflammation.At present,there is no research on angiogenesis in AS hip ligament.Objective: In this part of the experiment,we obtain ligaments tissues around AS and FNF hip joints in the process of surgery,and they were used as the experimental group and control group,respectively.The aim is to research whether the ligaments around the hip of the AS are accompanied with increased vascular regeneration.Method: Matrix degradation around vascular was the first step in angiogenesis.We examined the changes of expression of Fibronectin,Laminin,Collagen 1,Collagen 4,MMP1,MMP2,MMP9,TIMP1,TIMP2,TIMP3 through RT-PCR(Real Time-Polymerase Chain Reaction)technique to research the matrix degradation.CD31 is the surface marker of endothelial cells,Fibronectin and Laminin are able to reflect the matrix degradation around the vascular.So CD31 and Fibronectin,CD31 and Laminin double immunofluorescence have been used to demonstrate the matrix degradation,vascular density and morphological changes in hip joint ligament tissues of AS.Results: RT-PCR results showed that as compared with FNF hip ligament,the expression of MMP1,MMP2 and MMP9 in the hip ligament of AS patients increased by7.21 times,2.17 times and 2.19 times respectively,and Fibronectin,Laminin,Collagen1,Collagen4,TIMP1,TIMP2,TIMP3 expression decreased by 1.92 times,2.44 times,2.04 times,2.63 times,2.33 times,10.73 times,2.78 times respectively.The results of doubleimmunofluorescence showed that increased expression of CD31 and vascular density were observed in AS hip joint ligament,decreased expression of Fibronectin and Laminin were also observed in the AS hip joint ligament.These results indicated that matrix degradation was increased and angiogenesis was increased in AS hip joint ligament.Conclusion: Compared with FNF hip ligament tissue,matrix degradation and angiogenesis were increased in the AS hip joint ligament tissue.Part ?: Ang1 expression was increased in AS hip joint ligament tissues andprimary fibroblast,the expression of Ang1 and Tie2 increased with TNF-?stimulation in primary fibroblastsBackground: Many cytokines are involved in the process of angiogenesis,including vascular endothelial growth factor(VEGF),fibroblast growth factor,angiopoietins(Angs)and their receptors.The results of the gene chip prior to this study have confirmed that Ang1 in Angs family is up-regulated in AS hip ligament.Objective: In this part of experiment,we will further identify the expression of Ang1 ligand,Ang2 ligand,Tie2 receptor and CD31 marker moleuce in hip joint ligament tissue,and the expressions of Ang1,Ang2 in fibroblast of hip joint ligament tissues.TNF-? was used to stimulate primary fibroblast to future explore the causes of upregulation of Ang1 expression in AS.Method: We first examined the changes of Ang1,Ang2 and Tie2 expression in hip joint ligament tissues by RT-PCR at the level of gene and Western-Blot at the level of protein.Immunohistochemistry was used to semi-quantitative and location analysis expression of Ang1,Ang2 and Tie2.The expression of Tie2 was localized by CD31 and Tie2 double immunofluorescence.Secondly,after obtaining ligament tissue,the primary fibroblasts of AS and FNF were obtained by collagenase digestion,and primary fibroblasts were identified by CD90 and CD29.The changes of Ang1,Ang2 and Tie2 in fibroblasts were detected by RT-PCR and Western-Blot respectively at gene level and protein level.Ang1 and CD90 double immunofluorescence were used to detect the expression of Ang1 in fibroblasts and the localization of Ang1 was analyzed.The changes of Ang1 and Ang2 in supernatant of fibroblasts were detected by ELISA.Finally,in order to further investigate the upregulation of Ang1 expression in AS fibroblasts,we detected the expression of Ang1 ligand and Tie2 receptor after stimulating fibroblasts with TNF-?.Results: The results of RT-PCR showed that the expression of Ang1,Ang2 and Tie2 in the hip ligament of AS was 5.32 times,3.56 and 2.13 times higher than that of FNF.Western-Blot results suggest that increased expression of Ang1,Ang2 and Tie2 was observed in the hip ligament of AS.The gray values of Ang1(P <0.001),Ang2(P <0.001),Tie2(P <0.001)protein was significantly higher than that of FNF by quantitatively analyzed,Ang1 gray value / Ang2 gray value ratio is no difference.The results of immunohistochemistry showed that the expression of Ang1,CD31 and Tie2 protein in AS hip ligament were up-regulated,and Ang1 protein was expressed in the periphery of blood vessels and away from blood vessels,while Tie2 was mainly expressed around the blood vessels.CD31 and Tie2 double-labeled immunofluorescence results confirmed that Tie2 was expressed predominantly on endothelial cells.The results of primary fibroblast identification showed that 81.1% of the cells obtained by collagenase digestion were primary fibroblasts.The results of RT-PCR and Western-Blot showed that Ang1 was up-regulated at gene level and protein level in AS fibroblasts.The results of double-labeled immunofluorescence staining of CD90 and Ang1 suggest that the expression of Ang1 in the cytoplasm of AS fibroblasts is significantly higher than that of FNF.The results of ELISA showed that the mean concentration of Ang1 in AS culture medium was significantly higher than that in FNF(36.3 ± 5.08 ng/ml VS 17.06 ± 1.54 ng/ml,p<0.001).Ang1 was upregulated in a dose-dependent manner after stimulation of AS primary fibroblasts with TNF-?,and Tie2 receptor was also upregulated in a dose-dependent manner,however,this phenomenon was not found in FNF fibroblast.Conclusion: Compared with FNF,Ang1,Ang2 and Tie2,both gene level and protein level,increased in AS hip ligament tissue.Tie2 receptors were expressed in the AS hipligament tissue and were expressed predominantly in endothelial cells.Ang1,regardless of gene level or protein levels increased expression in AS fibroblasts,and in the cytoplasm synthesis and secretion to the extracellular,may be through the paracrine approach act on the endothelial cells in the ligament tissues.TNF-? can up-regulate the expression of Ang1 ligand and Tie2 receptor in AS hip ligament fibroblasts,which may be related to the inflammatory pathological changes of AS itself.Part ? AS fibroblast supernatant and COMP-Ang1 promote migration and tube formation of endothelial cellBackground: Angiogenesis is determined by the surrounding environment of the blood vessels.When the surrounding environment can not provide enough nutrients and oxygen for the tissue,the hypoxia tissue releases the angiogenic factor and promotes the formation of new blood vessels from the existing blood vessels.During the process of angiogenesis,the degradation of the perivascular matrix is the first step,which has been demonstrated in the first part of the experiment.Then the endothelial cells was stimulated by angiogenic factor and break out from the original blood vessels and jointly coordinate sptounting,branching and new lumenized network formation.Each new sprout will connect with surrounding sprouts through the tip cells,and ultimately the formation of a continuous lumen.Many genes synergetically promotes angiogenesis.In the process of angiogenesis,will COMP-Ang1 and supernatant of fibroblast promote endothelial cell migration and tubule formation?Objective: The purpose of this part of the experiment is to detect whether the Ang1 /Tie2 signaling system in the supernatant of AS and FNF fibroblasts can promote endothelial cell migration and tube formation,and use Ang1 recombinant protein COMP-Ang1 to detect whether it promotes endothelial cell migration and tubule formation to verify the role of Ang1 in promoting angiogenesis.Method:Ang1 / Tie2 signaling system can promote the migration of endothelial cells through scratch experiments.Angl / Tie2 signaling system can promote the formation ofvascular lumen in endothelial cells through in vitro tubule formation experiments.Results: The results showed that AS and FNF fibroblast conditioned medium could promote the migration of endothelial cells and tubule formation,and the role of AS fibroblast conditioned medium was more obvious.COMP-Ang1 promoted the migration and tubule formation of endothelial cells in a dose-dependent manner.When Tie2 receptor inhibitor was added to endothelial cell culture medium,the migration and tubule formation of endothelial cells were significantly inhibited.Indicating that Ang1/Ti2 signaling system can promote endothelial cell migration and tubule formation.Conclusion: Ang1 / Tie2 signaling system promotes angiogenesis by promoting endothelial cell migration and tube formation of endothelial cells.Part ? Ang1 mediates angiogenesis through Notch-1 pathwayBackground: Our experimental study has shown that Ang1 / Tie2 signaling system is highly expressed in AS hip ligaments and fibroblasts and promotes endothelial cell migration and tubule formation.However,Ang1 / Tie2 through what kind of signal pathway to promote angiogenesis is not entirely clear at present.Studies have shown that VEGF / Ang2 can promote angiogenesis through Notch-1 signaling pathway in synovial membranes of rheumatoid arthritis[1].Studies have also shown that alcohol promotes the vascular activity of endothelial cells through Notch-1 signaling pathway and Ang1-dependent approach [2].However,the correlation between the Ang1 / Tie2 signaling system and the Notch-1 signaling pathway in regulating angiogenesis was not researched.Objective: This part experiment was designed to investigate the relationship between the Ang1 / Tie2 signaling system and the Notch-1 signaling pathway,demonstrating that the Ang1 / Tie2 signaling system regulates endothelial cell migration and tubule formation through the Notch-1 signaling pathway.Method: First,whether COMP-Ang1 can activate the Notch-1 pathway.The expression of Notch-1 and its downstream target genes Hey1,Hey2 and Hes5 was detected by RT-PCR after different concentrations of COMP-Ang1 stimulation,and the expressionof Notch-1 receptor and DLL-4 ligand were also detected by Western-Blot.In addition,the Ang1 / Tie2 signaling system was blocked by adding the inhibitor of Tie2 receptor when adding COMP-Ang1 stimulation,after that,the expression of Notch-1 and its downstream genes Hey1,Hey2 and Hes5 were also detected by RT-PCR from the gene level.The expression of Notch-1 receptor and DLL-4 ligand were detected by Western-Blot protein level.Secondly,we will demonstrated the Ang1 / Tie2 signaling system regulated angiogenesis through the Notch-1 signaling pathway.The endothelial cells were stimulated with different COMP-Ang1 concentrations,and their effects on endothelial cell migration and tubule formation were examined.Notch-1 signaling pathway inhibitor ?-secretase inhibitor(DAPT)was added with COMP-Ang1 stimulation,and their effects on the migration and the tube formation of endothelial cells were examined.Results: The Notch-1 receptor and its DLL-4 ligand were up-regulated in endothelial cells in a dose-dependent manner after the different concentrations of COMP-Ang1 stimulated endothelial cells,and the expression of downstream target genes Hey1,Hey2 and Hes5 were also up-regulated.Different concentrations of COMP-Ang1 stimulated endothelial cells,meanwhile inhibiting Tie2 receptor,Notch-1 receptor and DLL-4 ligand expression did not change,the expression of downstream target genes Hey1,Hey2 and Hes5 also did not change.When endothelial cells were stimulated with different concentrations of COMP-Ang1,endothelial cell migration and tube formation increased.Different concentrations of COMP-Ang1 stimulated endothelial cells while inhibiting Notch-1 signaling pathway,endothelial cell migration and tube formation did not change significantly.Conclusion: The Ang-1 / Tie2 signaling system may regulate angiogenesis through the Notch-1 signaling pathway.